Chemokines and chemokine receptors are extensively and broadly involved in cancer metastasis. Previously, we demonstrated that epigenetic silencing of the chemokine CXCL12 sensitizes breast and colon cancer cells to endocrine signaling and metastasis to distant tissues. Yet, the precise mechanism whereby CXCL12 production by tumor cells regulates dissemination remains unclear. Here, we show that administration of CXCL12 extended survival of tumor-bearing mice by potently limiting metastasis of colorectal carcinoma or murine melanoma. Because secreted CXCL12 is a mixture of monomeric and dimeric species in equilibrium, oligomeric variants that either promote (monomer) or halt (dimer) chemotaxis were used to dissect the mechanisms interrupting carcinoma metastasis. Monomeric CXCL12 mobilized intracellular calcium, inhibited cAMP signaling, recruited β-arrestin-2, and stimulated filamentous-actin accumulation and cell migration. Dimeric CXCL12 activated G-protein-dependent calcium flux, adenylyl cyclase inhibition, and the rapid activation of ERK1/2, but only weakly, if at all, recruited arrestin, stimulated actin polymerization, or promoted chemotaxis. NMR analyses illustrated that CXCL12 monomers made specific contacts with CXCR4 that were lost following dimerization. Our results establish the potential for inhibiting CXCR4-mediated metastasis by administration of CXCL12. Chemokine-mediated migration and β-arrestin responses did not dictate the antitumor effect of CXCL12. We conclude that cellular migration is tightly regulated by selective CXCR4 signaling evoked by unique interactions with distinct ligand quaternary structures.

G-protein–coupled receptors (GPCRs) are key signaling molecules and are intensely studied. Whereas GPCRs recognizing small-molecules have been successfully targeted for drug discovery, protein-recognizing GPCRs, such as the chemokine receptors, claim few drugs or even useful small molecule reagents. This reflects both the difficulties that attend protein–protein interface inhibitor discovery, and the lack of structures for these targets. Imminent structure determination of chemokine receptor CXCR4 motivated docking screens for new ligands against a homology model and subsequently the crystal structure. More than 3 million molecules were docked against the model and then against the crystal structure; 24 and 23 high-scoring compounds from the respective screens were tested experimentally. Docking against the model yielded only one antagonist, which resembled known ligands and lacked specificity, whereas the crystal structure docking yielded four that were dissimilar to previously known scaffolds and apparently specific. Intriguingly, several were potent and relatively small, with IC ₅₀ values as low as 306 nM, ligand efficiencies as high as 0.36, and with efficacy in cellular chemotaxis. The potency and efficiency of these molecules has few precedents among protein–protein interface inhibitors, and supports structure-based efforts to discover leads for chemokine GPCRs.

Recently, we identified extracellular ubiquitin as an endogenous CXC chemokine receptor (CXCR) 4 agonist. However, the receptor selectivity and molecular basis of the CXCR4 agonist activity of ubiquitin are unknown, and functional consequences of CXCR4 activation with ubiquitin are poorly defined. Here, we provide evidence that ubiquitin and the cognate CXCR4 ligand stromal cell-derived factor (SDF)-1α do not share CXCR7 as a receptor. We further demonstrate that ubiquitin does not utilize the typical two-site binding mechanism of chemokine-receptor interactions, in which the receptor N terminus is important for ligand binding. CXCR4 activation with ubiquitin and SDF-1α lead to similar Gα_i-responses and to a comparable magnitude of phosphorylation of ERK-1/2, p90 ribosomal S6 kinase-l and Akt, although phosphorylations occur more transiently after activation with ubiquitin. Despite the similarity of signal transduction events after activation of CXCR4 with both ligands, ubiquitin possesses weaker chemotactic activity than SDF-lα in cell migration assays and does not interfere with productive entry of HIV-1 into P4.R5 multinuclear activation of galactosidase indicator cells. Unlike SDF-1α, ubiquitin lacks interactions with an N-terminal CXCR4 peptide in NMR spectroscopy experiments. Binding and signaling studies in the presence of antibodies against the N terminus and extracellular loops 2/3 of CXCR4 confirm that the ubiquitin CXCR4 interaction is independent of the N-terminal receptor domain, whereas blockade of extracellular loops 2/3 prevents receptor binding and activation. Our findings define ubiquitin as a CXCR4 agonist, which does not interfere with productive cellular entry of HIV-1, and provide new mechanistic insights into interactions between CXCR4 and its natural ligands.

Structural analysis of the interactions between a receptor and monomeric or dimeric forms of its ligand may aid in drug design.

The ability to interact with cell surface glycosaminoglycans (GAGs) is essential to the cell migration properties of chemokines, but association with soluble GAGs induces the oligomerization of most chemokines including CXCL12. Monomeric CXCL12, but not dimeric CXCL12, is cardioprotective in a number of experimental models of cardiac ischemia. We found that co-administration of heparin, a common treatment for myocardial infarction, abrogated the protective effect of CXCL12 in an ex vivo rat heart model for myocardial infarction. The interaction between CXCL12 and heparin oligosaccharides has previously been analyzed through mutagenesis, in vitro binding assays, and molecular modeling. However, complications from heparin-induced CXCL12 oligomerization and studies using very short oligosaccharides have led to inconsistent conclusions as to the residues involved, the orientation of the binding site, and whether it overlaps with the CXCR4 N-terminal site. We used a constitutively dimeric variant to simplify the NMR analysis of CXCL12-binding heparin oligosaccharides of varying length. Biophysical and mutagenic analyses reveal a CXCL12/heparin interaction surface that lies perpendicular to the dimer interface, does not involve the chemokine N terminus, and partially overlaps with the CXCR4-binding site. We further demonstrate that heparin-mediated enzymatic protection results from the promotion of dimerization rather than direct heparin binding to the CXCL12 N terminus. These results clarify the structural basis for GAG recognition by CXCL12 and lend insight into the development of CXCL12-based therapeutics.